1. Field of the Invention
The present invention relates to modified proteins or modified polypeptides which are used as carriers for carrying haptens (low molecular weight antigens) for the preparation of hapten antibodies or used as carriers for the preparation of polyvalent hapten antigens used in the immunoassay.
2. Description of Related Art
In the past, antibodies (Ab) for low molecular weight compounds acting as antigens, such as medicines or like chemicals, have been prepared by immunizing the immunorecipient animals with by inoculating conjugates which are prepared by combining these low molecular weight antigens (haptens) or homologues thereof with protein carriers, such as BSA (bovine serum albumin). While the carrier protein has functional groups including amino groups and carboxyl groups, it is considered that hapten molecules combine with the carrier protein at the site of amino or carboxyl groups. Hapten molecules combine more easily with amino groups.
However, all of the amino groups of a protein are not fully combined with hapten molecules to leave the steric structure inherent to the protein at the region where a number of uncombined amino groups are present so that the uncombined amino groups act as antigenic determinants (epitopes; Ag*) for the original protein. Accordingly, an antibody (Ab*) corresponding to the carrier protein is produced in the serum immunized with the protein carrying the hapten. In order to eliminate the influence of Ab*, the prepared anti-hapten anti-serum (Ab) must be absorbed by the carrier (Ag*) per se prior to use.
A similar inconvenience could occur when a hapten-protein conjugate is used in the passive agglutination immunoassay. In the passive agglutination immunoassay, plural mono-epitopic haptens each acting as a monovalent antigen (Ag) are carried by a carrier so that the composite of carrier and plural haptens carried thereby is allowed to act as a polyvalent antigen to cause matrix agglutination due to antigen-antibody binding. The carrier carrying plural hapten antigen (Ag) contacts with anti-hapten antibodies (Ab) so that an antigen-antibody matrix is formed. The amount of the formed matrix is detected by the determination of change in turbidity. When a free monovalent hapten antigen is present, formation of matrix is inhibited leading to reduction in turbidity. The amount of the free hapten antigen, i.e. the amount of the antigen contained in the sample, can be quantitatively determined by the analysis of the reduction in turbidity. Of course, when an antibody (Ab*) for the carrier (Ag*) is present, formation of matrix or agglutination occurs by the formation of Ag*-Ab* complex. Accordingly, it is necessary to absorb the used anti-hapten antibody (Ab) preliminarily by the carrier (Ag*) or to use in the passive agglutination immunoassay a composite which is formed by binding the hapten (Ag) to a carrier different from the carrier (Ag*) used in the immunization step.
One counterplan for obviating such inconvenience is to block all of the amino groups of the protein carrier so that the protein is fully denatured. However, realization of such counterplan is extremely difficult, and a large quantity of hapten is needed if such counterplan is adopted. Accordingly, this counterplan cannot be adopted when the quantity of hapten is small.
On the other hand, when the quantity of hapten is very small or the solubility of hapten in solvents is low to lower than the concentration of available hapten solution, it becomes impossible to introduce the hapten sufficiently into the carrier protein leading to insufficient titer of the resultant antibody. It is, therefore, desirable to increase the sites of the carrier at which hapten molecules are introduced so as to obtain a carrier into which the hapten is introduced at high efficiency.